The most effective way to fight cancer is at early stages, making early detection and risk assessment essential for patient survival. With recent advances in proteomics and metabolomics, there has been an explosion of newly discovered biomarkers indicative of cancer. However, detection of these biomarkers will require new diagnostic development that can be costly and take several years to develop. A universal diagnostic tool that can detect current and soon-to-be discovered cancer biomarkers is needed. Development of a new general diagnostic that can detect multiple cancer biomarkers in real-time is proposed. Recently a nucleic acid-based ribozyme, or aptazyme, capable of ligand-dependent exponential amplification was designed. The aptazyme has been shown to interchangeably detect theophylline and flavin mononucleotide (FMN) within physiological ranges. Unlike other diagnostics, such as RT-PCR, the system is isothermal and self-sustained which does not require other biological macromolecules and additional instrumentation. However, the current assay takes hours to perform, uses hazardous radioactive materials, and can detect only one ligand at a time. A new real-time, non-hazardous, and multiplexed aptazyme using fluorescence methods is proposed. Fvrster (fluorescence) resonance energy transfer (FRET) methods will be used to detect exponential amplification by fluorescently labeling the aptazyme. FRET will be coupled to activity of the aptazyme enabling real-time detection of ligand concentrations. As proof-of-principle, two aptazymes specific for cancer biomarkers prostate-specific antigen (PSA) and p21-activated kinase (PAK1) will be developed. Existing aptamer motifs for PSA and PAK1 will be incorporated into the aptazyme and each will be coupled to a unique FRET pair producing a distinct FRET signal for each ligand. The distinct FRET pairs will have minimal spectral overlap enabling multiplexed detection of ligands. Varying concentrations of PSA and PAK1 will be simultaneously assayed in the same sample in real-time. This system has the potential to quantitatively detect cancer biomarkers whether protein, small molecule, or nucleic acid. In addition, the ability to multiplex allows for individual assessment for each patient and monitoring patient response to treatment. Aptamers specific for new targets can be made with ease and the aptamer region within the aptazyme is interchangeable. The aptazyme system proposed here has the potential to bind any cancer biomarker and circumvents further rigorous assay development. PUBLIC HEALTH RELEVANCE: Detection of cancer biomarkers can reduce mortality rates in patients by facilitating individualized diagnosis and early treatment. It is likely that new biomarkers will be discovered, thus a general diagnostic tool that can detect all biomarkers (whether protein, small molecule, or nucleic acid) would circumvent timely and costly assay development.(1-4) The self-sustained system proposed in this application has the potential for universal and multiplexed detection of current and newly discovered cancer biomarkers in real-time.